ABSTRACT This MIRA application is to consolidate my effort on three separate GM grants: our long standing centrosome structure and microtubule nucleation grant (GM031627-30), yeast spindle pole body program project grant (Winey 1P01GM105537) and a MPI grant on the newly discovered phage tubulin PhuZ (Joe Pogliano, GM104556). This work is exciting and has been well appreciated. Most importantly, the collaborative efforts have been invaluable, resulting in remarkable synergies and a great expansion of our reach and understanding (genetics, in vivo function, computational modeling, single molecule biophysics). Similarly, our unique combina- tion of biophysics and structure has significantly enhanced what our collaborators could accomplish. Unfortu- nately under the new rules, I cannot continue my funded participation in these efforts. Tubulins play important and remarkably varied roles throughout eukaryotic and prokaryotic biology. In most eukaryotes, the microtubule (MT) cytoskeleton is responsible for chromosome segregation, nuclear positioning, vesicle trafficking and cell polarity. As the principle nucleator of MTs, the centrosome is of fundamental im- portance and harbors the centrioles, specialized structures required for cilia formation and contribute to spin- dle-pole positioning. The centrosome also has important roles beyond MT nucleation, including functions in cytokinesis, progression through the cell cycle, and in sequestering or positioning some proteins to control when and where they are active. Thus, the centrosome is important in embryonic development and human disease. While the analogous structure in the yeast S. cerevisiae (the spindle pole body or SPB) is morpholog- ically distinct, a conserved set of ?-tubulin complexes is used in both systems to nucleate MT assembly. Our long-term goals are to synthesize an atomic resolution picture of the relevant structural and functional in- teractions between ??- and ?-tubulin complexes, and to elucidate the principles underlying assembly and regu- lation. We have determined the structure of the yeast ?TuSC ring to 6.5 resolution, revealing principles of MT nucleation and protofilament number determination, and have identified at least two unexpected levels of regu- lation: via assembly mediated by oligomerization of the Spc110p attachment factor, and a closure event, lead- ing to activated nucleation. Proposed, we seek to expand the structural studies to atomic resolution by cryoEM and discover the role of PTMs and other factors in regulation. We will place in context via structures of the SPB and centrosome determined by cryoTomography, and better understand how the pericentrioloar material forms and uncover attachment factors that connect ?TuRCs to the Golgi. Our work on MT nucleation by ?-tubulin complexes has been complemented by our progress on the structure and biochemistry of prokaryotic tubulin homologs, including a ~3.3 structure of PhuZ:GMPCPP and ~4. of PhuZ:GDP, providing new insights into filament structure and general regulation by GTP hydrolysis. Here we focus on the novel PhuZ spindle and associated cell biology.